In this paper we describe the effects of over-expression of the Penicillium italicum gene encoding eburicol 14α-demethylase (cyp51), in Aspergillus niger strains with one or multiple copies of the gene encoding cytochrome P450 reductase (cprA), on the eburicol 14α-demethylase activity. Eburicol 14α-demethylase activity was determined by measuring the resistance of transformants against some eburicol 14α-demethylase inhibitors (DMIs). DMIs are widely used as fungicides in crop protection and human and veterinarian health care. DMI resistance in a transformant overexpressing both CPR and CYP51 was increased 5–30-fold compared to DMI resistance in the wild type strain, depending on the test compound used. Resistance in this strain was approximately 2–5-fold increased compared to DMI resistance in a transformant that was overexpressing the cyp51 gene but had only the wild type copy of the cprA gene and approximately 3–12-fold increased compared to a strain overexpressing the cprA gene (and having only the wild type copy of the cyp51 gene). These results show the importance of CPR overexpression for increasing cytochrome P450 activities in filamentous fungi.